Regulation of the Expression of the Hematopoietic Stem Cell Antigen CD34: Role of c-myb By Paola Melotti, De-Hui Ku, and Bruno Calabretta

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1 Bref Det~ntve Report Regulaton of the Expresson of the Hematopoetc Stem Cell Antgen CD34: Role of c-myb By Paola Melott, De-Hu Ku, and Bruno Calabretta From the Department of Mcrobolagy and Immunology, Jefferson Cancer Insttute, Thomas Jefferson Unversty, Phladelpha, Pennsylvana Summary The CD34 antgen defnes a subset of hematopoetc progentor cells wth self-renewal capacty and the ablty to reconsttute hematopoess n rradated prmates and marrow-ablated humans, but ts functon remans unknown. The c-myb protooncogene plays a fundamental role n hematopoess, most lkely va ts transcrptonal regulator functon. We report that c-myb proten transactvates the CD34 promoter va specfc nteracton wth multple Myb bndng stes n the 5' flankng regon of the gene and nduces expresson of the endogenous CD34 mtlna n rodent fbroblasts. Also, consttutve expresson of c-myb n CD34-negatve human globlastoma cells nduces expresson of CD34 mrna and synthess of the surface membrane antgen. These data drectly demonstrate that c-myb regulates the expresson of the hematopoetc stem cell antgen CD34 and rase the possblty that c-myb regulates hematopoess nducng a cascade of dfferentaton-related events. T he CD34 surface antgen s a hghly glycosylated transmembrane proten expressed on hematopoetc stem cells and lneage-specfc progentor cells, on a subset of bone marrow stromal cells and on small vessel endothelum of a varety of tssues (1-4). About 1-4% of normal bone marrow cells and ",,30% of blasts from acute leukema patents express CD34 (1, 2, 5). To date, CD34 remans the only welldefned human stem cell marker. CD34-postve cells have self-renewal capacty and ablty to reconsttute hematopoess n sublethally rradated prmates and marrow-ablated humans (6, 7). The functon of CD34 s not yet known, although ths proten has been proposed to play a role n hematopoetc cell adheson to stromal cells, perhaps facltatng the nteracton wth locally released growth factors, and to drectly functon as a sgnal transducer (3, 4, 8). The mechansms regulatng CD34 expresson n hematopoetc cells are not well understood, although there s evdence of transcrptonal and posttranscrptonal regulaton of CD34 expresson (9, 10). In lght of putatve Myb bndng stes present n the 5' flankng regon of the CD34 gene (9, 11), and the fundamental role of c-myb n hematopoetc cell prolferaton and/or dfferentaton (12-15), most lkely through the transcrpton regulator functon of Myb protens (16-19), we nvestgated the possblty that c-myb s drectly nvolved n transcrptonal regulaton of CD34 gene, possbly by nducng expresson of ts mrna and synthess of the surface membrane proten n nonexpressng cells. Materals and Methods Gel Retardaton Assay. HB101 cells contanng the parental pflag (IBI, New Haven, CT) expresson vector only or HB101 cells contanng the pc-myb Flag vector (20) were ncubated for 4 h n the presence of 1.5 mm sopropyl/~-d-thogalactopyranosde (IPTG) to an OD 600 of Myb expresson was determned n bacteral lysates by Western blot usng an ant-myb-specfc antbody (Upstate Botechnology Incorporated [UBI], Lake Placd, NY). 32p-labeled double-stranded probes were syntheszed by PCR and corresponded to three dfferent segments of the human CD34 5' flankng regon from -294 to -115; -132 to 54, and 67 to 234, based on the publshed sequence (9, 11). Gel retardaton assays were performed as descrbed (20). Transent Chloramt~hencol Acetyl-Transferase (CAT) Analyss. Constructs n whch dfferent CD34 promoter regons drve the bacteral CAT gene were prepared by PCR amplfcaton of human placental genomc DNA and by clonng the dfferent fragments of the CD34 5' flankng regon frst nto the pcrii vector (Invtrogen Corp., San Dego, CA) or drectly nto the puccat vector (Promega Corp., Madson, WI). CD34 SM-CAT and CD34 LM- CAT contan dentcal nucleotde substtutons n the Myb consensus sequence, from nucleotde 75 to 78 and 92 to 95 (CAAC to TGGC and GTTA to GCCC, respectvely), of the publshed sequence of the human CD34 gene (9, 11). TK-ts13 hamster fbroblasts were transfected, usng the calcum-phosphate precptaton method (21), wth 1 #g of CAT reporter plasmd wth or wthout 5 #g of effector plasmd (pmbl-dhfr, named psv myb, contanng the human c-myb cdna drven by SV40 early promoter) plus 1 /zg of a plasmd contanng the bacteral fl-galactosdase gene drven 1023 J. Exp. Med. 9 The Rockefeller Unversty Press ~ /94/03/1023/06 $2.00 Volume 179 March

2 by the DNA polymerase-c~ promoter, as an nternal control oftransfecton effcency. Cells were harvested 48 h after transfecton. Protens were extracted by freeze-thawng and normalzed for transfecton effcency usng the 3-galactosdase assay, as suggested by the manufacturer (Promega). Cellular lysates were ncubated wth [14C]chloramphencol and acetyl-coa for 1 h at 37~ Transactvaton of the reporter constructs was assayed by measurng the amount of acetylated [14C]chloramphencol by thn-layer chromatography followed by autoradography and scntllaton countng. Detecton of c-myb Proten n Transfected Cells. Levels of c-myb proten were determned n total cell extracts from 6 x 106 transfected TK-ts13 hamster fbroblasts and human globlastoma T98G cells by Western blot analyss wth a monoclonal ant-mouse c-myb antbody (UBI) and then wth a peroxdase labeled sheep ant-mouse Ig antbody (Amersham Corp., Arlngton Heghts, IL). Bound antbodes were revealed wth the Enhance Chemlumnescence Detecton System (ECL; Amersham Corp.). Expresson of CD34 mrna n Transfected Cells. TKots13 cells were transfected wth no plasmd, 5 #g of psv 3-gal contanng bacteral 3-galactosdase DNA under the control of the SV40 early promoter or 5/xg of psvmyb. RNA was extracted as descrbed (22) 24, 36, or 48 h after transfecton. Usng 0.7/~g of total RNA and 40 cycles CD34 mrna was amplfed by reverse transcrptase (RT)-PCR, as descrbed (23), wth a par of synthetc prmers correspondng to nucleotdes from 461 to 482 (5' prmer) and from 246 to 267 (3' prmer) of the publshed routne CD34 cdna sequence (24). Amplfed DNA was subjected to electrophoress, transferred to Zetabnd nylon flters (Cuno, Inc., Merden, CT) and detected by Southern hybrdzaton wth a y-[32p]atp end-labeled olgoprobe correspondng to nucleotdes 366 to 395 (24). In TKtsD cells consttutvely expressng c-myb, CD34 mr.na levels were also measured by RT-PCR usng 0.7/zg of total RNA and 40 cycles. In the dfferent T98G cell lnes CD34 mrna levels were also determned by RT-PCR. Prmers used correspond to nucleotdes 957 to 982 (5' prmer) and 1165 to 1190 (3' prmer); the olgoprobe ncludes nucleotdes 1109 to 1134, of the publshed human cdna sequence (25). Amplfcaton products were run on 1% agarose gel, blotted and probed wth the 32p end-labeled olgoprobe. CelISu~ace CD34 Expresson. Exponentally growng cells were harvested and ncubated (30 mn on ce n PBS contanng 0.1% gelatn, 0.01% sodum azde, 5 % fetal calf serum) wth antbodes to CD34 (mouse IgG1 ant-hpca1; Becton Dcknson & Co., Mountan Vew, CA), 32-mcroglobuln (BBM1) as postve controls, or CD16 (3G8, rrelevant IgG1), as negatve controls. Cells were washed and ncubated (30 mn on ce) wth FITC-conjugated goat ant-mouse Ig F(ab')2. Cells were washed and analyzed by flow-cytometry on a EPICS Profle Analyzer (Coulter Corp., Haleah, FL). Results c-myb Proten Interacts wth the 5' Flankng Regon of the CD34 Gene. To determne whether Myb nteracts wth putatve Myb bndng stes n the 5' flankng regon of the CD34 gene, gel retardaton assays were performed wth bacteral lysates contanng or not contanng Myb proten, and probed wth dfferent 32p-labeled DNA fragments of the CD34 5' flankng regon (Fg. 1). One retarded complex was detected n the lysate contanng Myb proten (Fg. 1, lanes 2, 5, and 8), but none n the lysate that lacked Myb proten (Fg. 1, lanes 3, 6, 9, and 12). No bndng was detected when a probe wth a 3-base substtuton n each of the two potental Myb Fgure 1. Myb proten bndng to CD34 5' flankng regon. Lanes 1, 4, 7, and I0, free probe only; lanes 3, 6, 9, and 12, probe plus 1/xg of parental bacteral lysate; lanes 2, 5, 8, and 11, probe plus 1 #g of bacteral lysate contanng the human c-myb proten. The probe used n lanes contans three nucleotde substtutons n both Myb bndng stes. The dfferent regons of the CD34 promoter used as probe are ndcated on the top. consensus sequences was used (Fg. 1, lane tt), demonstratng the specfcty of the nteractons. CD34 Promoter Actvty Is 7~ansactvated by c-myh To determne whether c-myb transactvates the CD34 5' flankng regon, constructs n whch dfferent fragments of the CD34 promoter drve bacteral CAT gene expresson were prepared by clonng the segments correspondng to nucleotdes -666 to 234 (CD34 L-CAT), -132 to 234 (CD34 M-CAT), and 31 to 187 (CD34 S-CAT) (9, 11) nto the puccat vector (Fg. 2 A), and transfected nto wld-type TK-ts13 Syran hamster fbroblasts or n cells consttutvely expressng c-myb (SV-mybTK-ts13). TK-ts13 cells were transfected at a 5:1 effector-to-reporter rato wth plasmd pmbm-dhfr. Ths plasmd, whch contans the human c-myb cdna under the control of the SV40 early promoter and enhancer lnked to the dehydrofolate reductase codng sequence for methotrexate selecton (11) and the descrbed above reporter plasmds, were used n the experments. Levels of CAT actvty were assayed 48 h later (Fg. 2 B). c-myb nduced 8-, 10-, and 14-fold ncreases, respectvely, n CAT expresson drven by the CD34 L-CAT (Fg. 2 B, lane 3), CD34 M-CAT (Fg. 2 B, lane 5), and CD34 S-CAT (Fg. 2 B, lane 7) CD34 5' flankng regon segments. Transactvaton was abolshed when mutatons were ntroduced n the Myb bndng stes of the CD34 S-CAT reporter vector (CD34 SM-CAT; Fg. 2 B, lane 9). To determne whether the Myb bndng stes not ncluded n the segment correspondng to CD34 S-CAT (nucleotdes 31 to 187) are also nvolved n CD34 5' flankng regon transactvaton, TK-ts13 and SVmybTK-ts13 cells were transfected wth CD34 LM-CAT contanng the 900-bp fragment of the CD34 5' flankng regon wth mutatons n the two most proxmal Myb bndng stes (9, 11). Transactvaton was unaffected (Fg. 2 B, lane 4), compared wth the wld-type L-CAT construct (Fg. 2 C, bar 2), ndcatng that the more dstal Myb consensus sequences are also functonal bndng stes. CAT assays were also performed usng TK-ts13 or SVmybTK-ts13 cells transfected wth CD34 L-CAT and CD34 LM-CAT n the presence of an excess (100:1, molar rato) of a 36-bp fragment of the CD34 5' flankng regon (nucleotdes 31 to 67) contanng two wld-type or two mutated Myb bndng stes Role of c-myb n Hematopoetc Stem Cell Antgen CD34 Expresson

3 of CAT actvty were dentcal n TK-ts13 cells transfected wth psvcat n the presence or absence of an excess (100:1 molar rato) of compettor (not shown). CD34 Expresson Is Regulated by c-myt~ To test the predcton, from the above results, that c-myb may upregulate CD34 expresson, RT-PCR analyss of CD34 mrna expresson was performed n TK-ts13 cells 24, 36, and 48 h after transfecton wth psvmyb. CD34 mrna was detectable only n c-myb-expressng cells (Fg. 3 A, lanes 3-5), but not n mocktransfected TK-ts13 cells (Fg. 3 A, lane 1), or n TK-ts13 cells transfected wth the psv3-gat plasmd (Fg. 3 A, lane 2) used as controls. In addton, CD34 mrna expresson was detected n SVmyb TK-ts13 cells consttutvely expressng c-myb proten (Fg. 3 B, rght, lane 2). Antbodes are avalable for detecton of human, but not rodent CD34 protens; therefore, to prove that nduced CD34 mrna expresson results n synthess and expresson of the encoded proten, human globlastoma T98G cells (nonexpressng c-myb) were transfected wth a c-myb cdna and assayed for Myb proten and CD34 expresson. Western blot analyss revealed the presence of a 75-kD proten correspondng to c-myb, n SVmyb T98G cells (Fg. 4 A, lane 2), n SVmybM T98G cells selected wth ncreasng concentratons of methotrexate (fnal concentraton 22 #M) to nduce amplfcaton Fgure 2. c-myb transactvaton of CD34 5' flankng regon n Tk-ts13 hamster fbroblasts. (A) Constructs n whch dfferent CD34 promoter regons drve the bacteral CAT gene. (B) Autoradograms of CAT actvty n lysates of TK-ts13 transfected wth the negatve control puccat (lane 1), CD34 DCAT (lane 2), CD34 M-CAT (lane 4), CD34 S-CAT (lane 6), CD34 SM-CAT (lane 8), or cotransfected wth psv-myb (lanes 3, 5, 7, and 9, respectvely). (C') Scntllaton countngs of acetylated [14C]chloramphencol n lysates of: TK-ts13 (lane I), and SVmyb-TK-ts13 cells (lane 2) transfected wth CD34 I~CAT; TK-ts13 (lane 3) and SV-Myb TK-ts13 cells (lane 4) transfected wth CD34 LM-CAT, TK-ts13 (lane 5), and SVmyb TK-ts13 cells (lane 6) transfected wth CD34 L-CAT plus a synthetc compettor contanng two wld-type Myb bndng stes; TK-ts13 (lane 7) and SVmyb TK-ts13 cells (lane 8) transfected wth CD34 LM-CAT plus a synthetc compettor contanng two wld-type Myb bndng stes; TK-ts13 (lane 9) and SVmyb TK-ts13 cells (lane 10) transfected wth CD34 L-CAT plus a synthetc compettor contanng two mutated Myb bndng stes; and TK-ts13 (lane 11) and SVmyb TK-ts13 cells (lane 12) transfected wth CD34 LM-CAT plus a synthetc compettor contanng two mutated Myb bndng stes. Transactvaton of CD34 L-CAT and CD34 LM-CAT was abolshed by the wld-type compettor (Fg. 2 C, bars 6 and 8) but was unaffected by the mutated olgomer (Fg. 2 C, bars 10 and 12), further demonstratng that the transactvaton of the CD34 promoter depends drectly on c-rnyb expresson and nteracton wth Myb bndng stes. The 36-bp compettor contanng two Myb bndng stes was not toxc, as levels of CAT actvty were dentcal n TK-ts13 cells transfected Fgure 3. CD34 mrna levels n TK-ts13 cells transfected wth psvmyb. (A) Expresson of CD34 mrna n transently transfected TK-ts13 cells. (Lane 1) cells transfected wth no plasmd; (lane 2) cells transfected wth 5/zg ofpsv 3-gal; (lanes 3-5) cells transfected wth 5 #g of psvmyb. KNA was extracted as descrbed (22) 48 h after transfecton except n lanes 3 and 4 (RNA extracted 24 and 36 h after transfecton, respectvely). To exclude amplfcaton from genomc DNA, RT-PCR reactons were done n the absence of reverse transcrptase (-). In controls, reactons were also performed n the absence of RNA. Endogenous ~-actn mrna levels were also measured to ensure that a smlar amount of RNA was analyzed for CD34 mp, NA expresson. (B) Expresson of CD34 mrna n TKts13 cells consttutvely expressng c-myb. (Left) levels of c-myb proten determned by Western blot analyss. (Lane 1) untransfected TK-ts13 cells; (lane 2) SVmybTK-ts3 cells; (lane 3) control HL-60 cells. (Rght) CD34 mrna levels determned by RT-PCR n untransfected TK-ts13 (lane 1) and n SV-myb TK-ts13 cells (lane 2) n the presence (+) or absence (-) of reverse transcrptase; (lane 3) P,T-PCR reacton n the absence of RNA n the reacton mxture Melott et al. Bref Defntve Report

4 rr UJ m Z --t W o w A #.a os,6 /, W Fgure 4. CD34 mrna levels n T98G human globlastoma cells consttutvely expressng c-myb. (.4) Levels of c-myb proten were determned by Western blot analyss n untransfected T98G cells (lane I), n SV-mybtransfected T98G cells (lane 2), n SV-myb T98G cells after methotrexate selecton (SVmyb M T98G) (lane 3), and n the control MEL cells (lane 4). (B) CD34 mrna levels were determned by RT-PCR n the presence (+) or absence (-) of reverse transcrptase. Each lane s representatve of three ndependent experments wth smlar results. (Lane 1) negatve control, HL-60 cells; (lane 2) T98G; cells, (lane 3) SV-myb T98G cells; (lane 4) SV-myb T98G cells after methotrexate selecton; (lane 5) postve control, KG-la cells (only one-ffth of the reacton was used); (lane 6) RT- PCR reacton n the absence of RNA n the reacton mxture. of the c-rnyb transcrpton unt (Fg. 4 A, lane 3), but not n parental T98G cells (Fg. 4 A, lane 1). In SVmybM T98G cells, levels of c-myb proten expresson were at least sevenfold greater than n SVmyb T98G cells, as revealed by denstometrc scannng of the flm (Fg. 4 A, compare lanes 3 and 4). Expresson of CD34 mrna was not detected n control HL-60 cells (Fg. 4 B, lane I) or n nontransfected T98G cells (Fg. 4 B, lane 2), but was found n SVmyb T98G (Fg. 4 B, lane 3), and, at hgher levels, n SVmybMT98G cells (Fg. 4 B, lane 4). Phenotypc analyss performed to montor surface expresson of the human CD34 antgen on T98G, SVmyb T98G and SVmybMT98G cells (Fg. 5 B) ndcated expresson of CD34 antgen on a small proporton (4.3 +_ 1.2%) of T98G cells. Low densty CD34 expresson was detected on SVmyb T98G cells, and most SVmybMT98G expressed CD34 at a varable level, wth average densty approxmately half of that detected n KG-la myeloblastc leukema cells used as postve control (Fg. 5 A). Dscusson The role of c-myb n hematopoess lkely derves from ts transactvatng functon, but the search for the relevant target(s) remans elusve, snce two hematopoetc-assocated targets, the MIM-1 and the lysozyme genes dentfed n avan cells, are expressed at a relatvely late stage of myelod dfferenta- ".,t 0r j. "..~ lll~'~ ~ I,,,lllll~ J I I'.~ I0-~ 10o FLUORESCENCE INTENSITY, Ioglo Fgure 5. Cell surface expresson of CD34 n T98G consttutvely expressng c-myb. (A) KG-la cells:..., negatve control; -, ant-cd34. (B) -, T98G; ant-cd34; - - -, SVmyb T98G; ant-cd34,...-, SVmybMT98G; ant-cd34,.-., negatve control analyss, attached for the three cell lnes, s reported, x-axs, number of cells; y-axs, fluorescence ntensty, log scale. The analyss was repeated twce wth dentcal results. ton. In contrast, expresson of the CD34 stem cell marker declnes durng late stages of myelod dfferentaton and maturaton. Our data suggest that CD34 represents a prmary target of c-myb regulaton durng the earlest recognzable stages of hematopoetc progentor cell commtment and dfferentaton. A temporal relatonshp n whch c-myb expresson precedes that of CD34 n human stem and progentor cells s dffcult to establsh because prmtve CD34-postve cells express c-myb, and CD34-negatve stem cells, f they exst, cannot be dentfed yet. A functonal and temporal relatonshp between c-myb and CD34 expresson mght be explored durng the dfferentaton of embryonc stem cells. In these cells a stage can be dentfed at whch c-myb, but not CD34, s expressed (26); ths observaton may support the nvolvement of c-myb n regulatng CD34 expresson durng embryonal hematopoetc development. Our data clearly ndcate that c-myb s a transcrptonal factor nvolved n regulatng CD34 expresson, but do not exclude that others are, too. Bndng stes for ets protens are present n the CD34 5' flankng regon, some n close proxmty wth Myb bndng stes, and prelmnary fndngs suggest that c-myb and ets-2 act synergstcally to transactvate the CD34 promoter. In addton, t s lkely that c-myb expresson s the only prerequste for CD34 expresson, as mpled by the lack of CD34 expresson n the HL-60 cells promyelocytc leukema cells that express c-myb at hgh levels Role of c-myb n Hematopoetc Stem Cell Antgen CD34 Expresson

5 KG-la and HL-60 represent relatvely close stages of myelod dfferentaton and express c-myb at hgh levels, and yet only KG-la cells express CD34. Ths suggests ether postve regulaton of CD34 expresson by a c-myb partner n KG-la cells, or negatve regulaton n HL-60 cells by a suppressor of CD34 expresson. In ths regard, a functonal bndng ste for the myelod-specfc transactvator NF-M contguous to a c-myb bndng ste appears to be necessary for c-myb regulaton of the MIM-1 promoter (27); a smlar mechansm may operate to control CD34 expresson n early progentor cells. Fnally, the ncreased fracton of CD34-postve cells n acute leukema patents correlates wth the enhanced clonogenc potental of these cells and ther hgh levels of c-myb expresson. In conjuncton wth such observatons, our data on c-myb regulaton of CD34 expresson mght provde nsghts nto the couplng of dfferentaton arrest and growth advantage n leukemc cells. We wsh to thank Davd Dcker for performng the FACS analyss and Dr. Bce Perussa for crtcal readng of the manuscrpt. P. Melott was supported by a fellowshp of the Assocazone Italana Rcerca sul Cancro (AIRC). D.-H. Ku was supported by tranng grant 1T32 CA from the Natonal Insttutes of Health (NIH). B. Calabretta s a scholar of the Leukema Socety of Amerca. Ths work was supported n part by NIH (CA-46782) and Amercan Cancer Socety (CH455A and CH492) grants to B. Calabretta. Address correspondence to Dr. Bruno Calabretta, Department of Mcrobology and Immunology, Jefferson Cancer Insttute, Bluemle Lfe Scences Buldng, Thomas Jefferson Unversty, 233 South 10th Street, Phladelpha, PA P. Melott s on leave from the Department of Pedatrcs, Unversty of Verona, Verona, Italy Receved for publcaton 1 November 1993 and n revsed form I December References 1. Cvn, C.I., L.C. Strauss, C. Brovall, M.J. Fackler, J.F. Schwartz, and J.H. Shaper Antgenc analyss of hemopoess. III. Hematopoetc progentor cells surface antgen defned by a monoclonal antbody rased aganst KGla cells, j. Immunol. 133: Katz, F.E., lk. Tndle, D.K. Sutherland, and M.F. Greaves Identfcaton of a membrane glycoproten assodated wth haemopoetc progentor cells. Leuk. Res. 9: Dela, D., M.G. Lampugnan, M. Resnat, E. Dejana, A. Aello, E. Fontanella, D. Solgo, M.A. Perott, and M.F. Greaves CD34 expresson s regulated recprocally wth adheson molecules n vascular endothelal cells n vtro. Blood. 81: Smmons, P.J., and B. Torok-Storb CD34 expresson by normal precursors n normal human adult bone marrow. Blood. 78: Tndle, R.W., R.A.B. Nchols, L. Chan, D. Campana, D. Catovsky, and G.D. Brne A novel monoclonal antbody BI-3C5 recognzes myeloblasts and non-b non-t lymphoblasts n acute leukemas and CGL blast crses, and reacts wth mmature cells n normal bone marrow. Leuk. Res. 9:1. 6. Berenson, K.J., R.G. Andrews, W.I. Bensnger, D. Kalamasz, G. Kntter, C.D. Buckner, and I.D. Bernsten Antgen CD34 marrow cells engraft lethally rradated baboons. J. Cln. Invest. 81: Berenson, R.J., W.I. Bensnger, R.S. Hll, R.G. Andrews, J. Garca-Lopez, D.F. Kalamasz, B.J. Stll, G. Sptzer, C.D. Buckner, I.D. Bemsten, and E.D. Thomas Engraftment after nfuson of CD34 + marrow cells n patents wth breast cancer or neuroblastoma. Blood. 77: Fackler, M.J., C.I. Cvn, D.R. Sutherland, M.A. Baker, and W.S. May Actvated proten knase C drectly phosphory- lates the CD34 antgen on hematopoetc cells. J. Bol. Chem. 265: Burn, T.C., A.B. Satterthwate, and D.G. Tenen The human CD34 hematopoetc stem cell antgen promoter and a 3' enhancer drect hematopoetc expresson n tssue culture. Blood. 80: Satterthwate, A.B., K. Borson, and D.G. Tenen Regulaton of the gene for CD34, a human hematopoetc stem cell antgen, n KG-1 cells. Blood. 75: He, X.-Y., V.P. Antao, D. Basla, J.C. Marx, and B.K. Davs Isolaton and characterzaton of the human CD34 gene. Blood. 79: Clarke, M.F., J.F. Kukowska-Latallo, E. Westn, M. Smth, and E.V. Prochownck Consttutve expresson of a c-myb cdna blocks Frend routne erythroleukema cell dfferentaton. Mol. Cell. Bol. 8: Todokoro, K., K.J. Watson, H. Hgo, H. Amanuma, H. Kuramoch, H. Yanagsawa, and Y. Ikawa Down regulaton ofc-myb gene expresson s a requste for erythropoetnnduced erythrod dfferentaton. Pro~ Natl. Acad. Sc. USA. 85: Gewrtz, A.M., and B. Calabretta A c-myb antsense olgodeoxynucleotde nhbts normal human hematopoess n vtro. Scence (Wash. DC). 242: Mucensk, M.L., K. McLan, A.B. Ker, S.H. Swerdlow, C.M. Schrener, T.A. Mller, D.W. Petryga, W.J. Scott, and S.S. Potter A functonal c-myb gene s requred for normal mouse fetal hepatc hematopoess. Cell. 65: Bedenkapp, H., V. Borgmeyer, A.E. Sppel, and K.-H. Klempnaner Vral myb oncogene encodes a sequence-specfc DNA-bndng actvty. Nature (Lond.). 335: Melott et al. Bref Defntve Report

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